Anti-glycoprotein VI scFv fragment for treatment of thrombosis

ABSTRACT

The present invention relates to a single chain variable fragment (scFv 9O12.2) directed against human glycoprotein VI, constituted of the VH and VL domains of 9 O12.2.2 monoclonal antibody linked via a (Gly 4 Ser) 3  peptide and followed by a “c-myc” flag, represented by SEQ ID NO:1. The invention also concerns functional variants of said scFv 9O12.2 fragment with identical heavy and light chains complementary determining regions 1, 2 and 3, and preferably humanized functional variants such as the humanized scFv fragments represented by SEQ ID NO:28 or SEQ ID NO:47. The invention also relates to nucleic acids encoding such a scFv fragment, expression vectors and host cells to produce such a scFv fragment, as well as therapeutic uses of such a scFv fragment.

TECHNICAL FIELD

The present invention relates to a single chain variable fragment (scFv9O12.2) directed against human glycoprotein VI, constituted of the VHand VL domains of 9O12.2 monoclonal antibody linked via a (Gly₄Ser)₃peptide and followed by a “c-myc” flag, represented by SEQ ID NO:1. Theinvention also concerns functional variants of said scFv 9O12.2 fragmentwith identical heavy and light chains complementary determining regions1, 2 and 3, and preferably humanized functional variants such as thehumanized scFv fragments represented by SEQ ID NO:28 or SEQ ID NO:47.The invention also relates to nucleic acids encoding such a scFvfragment, expression vectors and host cells to produce such a scFvfragment, as well as therapeutic uses of such a scFv fragment.

BACKGROUND ART

Acute coronary and cerebrovascular accidents are currently the firstdeath cause in the world. In addition, the global incidence ofrecurrence and death in the 6 month post-treatment period after an acutecoronary syndrome is still 8-15%. In the case of acute coronary syndromewith ST segment elevation, mechanical treatment with coronaryangioplasty and introduction of a stent is highly efficient to urgentlyrestore coronary artery flow, but does not prevent morbidity/mortalityfor about 15% of patients in the next 6 months.

Thrombolytic treatments, which are based on long term fibrinolytic,anticoagulant and anti-aggregating drugs associations, give even lessencouraging results. Indeed, despite improvements in medical treatmentof thrombosis, morbidity/mortality at 6 months is similar to thatobserved for acute coronary syndrome without segment ST elevation.

Concerning cerebrovascular ischemic accidents, treatments are still verylimited due to the generally late caring of most patients and to thehemorrhagic risk of currently available anti-thrombotic treatments.

There is thus a real pressing clinical need for improving treatments forcardiovascular diseases, and especially for new molecules with improvedfeatures compared to available molecules, in particular for moleculeswith a reduced hemorrhagic effect.

Platelets-collagen interactions are critical in the appearance of acutearterial thrombosis and post-thrombotic vascular remodeling.Glycoprotein VI (GPVI), the main receptor for platelets activation bycollagen, has been demonstrated in animals to play a role inexperimental thrombosis, vascular remodeling, atherothrombosis and acutemyocardial ischemia.

Contrary to αIIbβ3 integrin antagonists, which are currently used inthrombosis treatment and inhibit platelets final activation phase, GPVIis implicated into platelets initial activation phase, and GPVIantagonists should thus prevent not only platelet aggregation, but alsosecondary agonists liberation as well a growth factors and cytokinessecretion resulting into vascular lesions development. In addition, GPVIdeficit is not associated with a high hemorrhagic risk, which is acrucial feature for patient's safety. Finally, GPVI expression islimited to platelets, and thus represents a perfectly specific targetfor anti-thrombosis treatment.

GPVI antagonists should thus be efficient for specifically andefficiently preventing primary or secondary thrombosis, while involvingonly a low hemorrhagic risk.

Various kinds of potential GPVI antagonists have been generated. In oneapproach, a soluble GPVI recombinant protein has been generated, whichis a fusion protein between GPVI extracellular domain and human Ig Fcdomain (see for instance WO 01/00810 (1) and WO 03/008454 (2)). Thussoluble recombinant GPVI protein competes with platelet GPVI for bindingcollagen. Encouraging results were first obtained with this soluble GPVIprotein in a thrombosis murine model (3), but these results were notconfirmed (4). In addition, this approach involves structural,functional and pharmacological disadvantages. First, this compound is ahigh molecular weight protein (˜160 kDa) the half life of which isexpected to be short. Since GPVI contains at least one cleavage site forproteases, the hydrolysis of the soluble recombinant GPVI-Fc protein hasto be envisaged. When bound to collagen, the protein will expose its Fcdomain to the blood stream. Human platelets (but not mice platelets) andleucocytes express the low affinity Fc receptor (FcγRIIA) at theirsurface. Cross-linking of the platelet FcγRIIA by immobilized GPVI-Fc issusceptible to activate platelets and thus to have an opposite effect tothe one expected. The timing and the dose to which the protein should beadministrated also cause problem. Once bound to collagen platelets arerapidly and irreversibly activated. Thus, to be effective GPVI-Fc shouldbe administrated before platelet activation that is before thethrombotic event, a situation rare in current medicine. Furthermore, theamount of protein that should be administrated will vary as a functionof the size and the nature of the vascular lesion, a parameterimpossible to predict.

Many others have tried to develop neutralizing monoclonal antibodiesdirected against human GPVI. For instance, EP 1224942 (5) and EP 1228768(6) disclose a monoclonal anti-GPVI antibody JAQ1, which specificallybinds to mouse GPVI, for the treatment of thrombotic disease. JAQ1antibody induces irreversible internalization of the GPVI receptor onmouse platelets.

EP1538165 (7) describes another monoclonal anti-GPVI antibody hGP 5C4,which Fab fragment was found to have marked inhibitory effects on themain physiological functions of platelets induced by collagenstimulation: stimulation of collagen-mediated physiological activationparameters PAC-1 and CD 62P-Selectin was completely prevented by hGP 5C4Fab, and hGP 5C4 Fab potently inhibited human platelet aggregation exviva without any intrinsic activity.

WO 2005/111083 (8) describes 4 monoclonal anti-GPVI antibodies OM1, OM2,OM3 and OM4, that were found to inhibit GPVI binding to collagen,collagen-induced secretion and thromboxane A2 (TXA2) formation in vitro,as well as ex vivo collagen-induced platelet aggregation afterintravenous injection to Cynomolgus monkeys. OM4 also appears to inhibitthrombus formation in a rat thrombosis model.

WO 01/00810 (1) also describes various monoclonal anti-GPVI antibodiesnamed 7I20.2, 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, IP10.2, 4L7.3,7H4.6, 9O12.2, 7H14.1, and 9E18.2, as well as several scFv fragmentsnamed A9, A10, C9, A4, C10, B4, C3 and D11. Some of these antibodies andscFv fragments were found to inhibit GPVI binding to collagen, includingantibodies 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, IP10.2, 4L7.3, 7H4.6,and 9O12.2, and scFv fragments A10, A4, C10, B4, C3 and D11.

In addition, 9O12.2 Fab fragments were found to completely blockcollagen-induced platelet aggregation and secretion, to inhibit theprocoagulant activity of collagen-stimulated platelets and plateletadhesion to collagen in static conditions, to impair platelet adhesion,and to prevent thrombi formation under arterial flow conditions (9).

However, none of the currently known anti-GPVI antibodies have provenreally efficient in vivo for preventing and/or treating cardiovasculardiseases implicating platelet aggregation such as arterial and venousthrombosis, restenosis, acute coronary syndrome, and cerebrovascularaccidents due to atheroscleroris. Until recently the different anti-GPVIantibodies that have been reported appeared not fitted for thedevelopment of an antithrombotic for medical use in human. Only fewantibodies have been reported to have inhibitory properties. This is thecase of JAQ1 that is directed to mouse GPVI and does not cross reactwith human GPVI (10). Human scFvs directed to human GPVI have also beenreported to be inhibitory (11,12) but their affinity appears to be low.Very recently, new inhibitory antibodies with a good affinity for humanGPVI have been characterised (13) and proposed to be developed astherapeutic tools.

However, cross-linking of GPVI at the platelet surface by a divalent ormultivalent ligand results in platelet activation. This is the case ofthe 9O12.2 IgGs that activate platelets via GPVI dimerisation and viacross-linking of GPVI to the low affinity Fc receptor (FcγRIIA) (9).Fab′₂ also activate platelets via GPVI dimerisation (9). In contrast,monovalent 9O12.2 Fab fragments are inhibitory. However these fragmentscould not be used in therapeutic due to their size and their animalorigin which makes them immunogenic in human patients.

There is thus still a need for an efficient neutralizing GPVIantagonist, which would inhibit with high efficiency the initial phaseof platelets aggregation, with a low hemorrhagic risk, as well as a lowimmunogenic effect.

DISCLOSURE OF THE INVENTION

From the 9O12.2 monoclonal anti-GPVI antibody hybridoma, the inventorshave generated a single chain variable fragment (scFv) 9O12.2 which hasthe advantage to contain the functional inhibitory motif in a fragmentof reduced size without any immunogenic constant domains (CH₁ and CL).The scFv is the starting material to produce fragments with an improvedimmune tolerance and stability in order to obtain a format adapted to atherapeutic use.

Technologic approaches to design such fragments have previously beenreported in details, however there are many difficulties to overpassmaking each scFv construct a specific case. These difficulties includethe cloning of antibody V-genes by bypassing aberrant transcripts (14),identification of a linker well-suited for correct folding, associationof V-domains into a monomeric scFv and finally the selection of aprocaryotic expression system that enable expression of the scFv in afunctional soluble form.

The invention thus concerns a single chain variable fragment directedagainst human glycoprotein VI, comprising:

-   -   a VH domain comprising CDR1, CDR2 and CDR3 regions constituted        of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4,    -   a peptide linker, and    -   a VL domain comprising CDR1, CDR2 and CDR3 regions constituted        of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In a preferred embodiment, said scFv fragment according to the inventionfurther comprises a peptide tag, useful for purification, and optionallya short peptide spacer between the core scFv fragment (comprising the VHand VL domains and the peptide linker) and the peptide tag.

An antibody is a roughly Y-shaped molecule composed of two differentpolypeptide chains named “heavy” and “light” chains, an antibody beingconstituted of two heavy and two light chains linked by disulfide bonds.Each heavy chain is composed of a variable domain (“VH domain”) and 3constant domains (“CH1”, “CH2” and “CH3” domains), while each lightchain is composed of a variable domain (“VL domain”) and a constantdomain (“CL domain”).

A “single chain variable fragment” or “scFv fragment” refers to a singlefolded polypeptide comprising the V_(H) and V_(L) domains of an antibodylinked through a linker molecule. In such a scFv fragment, the V_(H) andV_(L) domains can be either in the V_(H)-linker-V_(L) orV_(L)-linker-V_(H) order. In addition to facilitate its production, ascFv fragment may contain a tag molecule linked to the scFv via aspacer. A scFv fragment thus comprises the V_(H) and V_(L) domainsimplicated into antigen recognizing but not the immunogenic constantdomains of corresponding antibody.

In addition, each variable domain (VH or VL domain) is composed of 4“framework regions” (FR1, FR2, FR3 and FR4), which display lessvariability among antibodies and are involved in the formation of βsheets forming the structural framework of the variable domain, and of 3hypervariable regions commonly named “complementary determining regions”1, 2 and 3 (CDR1, CDR2, CDR3), which correspond to 3 loops juxtaposed inthe folded variable domain at the edge of each β sheet. The 3 CDRregions are crucial for determining an antibody or antibody fragmentspecificity since they are the part of the variable domain mainly incontact with the antigen, especially the CDR3 region of each chain,which corresponds to the rearranged region of the heavy and light chainsand is even more variable and more directly in contact with the specificantigen.

Thus, the invention encompasses all functional variants of scFv 9O12.2which keep identical CDR regions. The scFv fragment 9O12.2 CDR regionsdata are presented in following Table 1:

TABLE 1scFv 9O12.2 CDR regions. CDR regions were identified using Kabat andChotia nomenclature (15, 16) Position in scFv CDR 9O12.2 (aa) SequenceVH CDR1 26-35 GYTFTSYNMH (SEQ ID NO: 2) VH CDR2 50-66GIYPGNGDTSFNQKFKG (SEQ ID NO: 3) VH CDR3 99-109GTVVGDWYFDV (SEQ ID NO: 4) VL CDR1 159-174RSSQSLENSNGNTYLN (SEQ ID NO: 5) VL CDR2 190-196 RVSNRFS (SEQ ID NO: 6)VL CDR3 229-237 LQLTHVPWT (SEQ ID NO: 7)

By a “peptide linker” is meant a flexible peptide that permits anappropriate folding of the scFv fragment, ie an appropriate folding ofthe VH and VL domains and their capacity to be brought together. Inaddition, such a peptide linker should permit folding into a monomericfunctional unit. When the scFv is assembled in the VH to VL orientation(V_(H)-linker-V_(L)), an scFv with a linker of 3 to 12 residues cannotfold into a functional Fv domain and instead associates with a secondmolecule to form a bivalent dimer. Reducing below 3 residues leads totrimers. In this case, a suitable linker should thus have at least 12and preferably less than 25 aminoacids, preferably between 14-18, 14-16,or 15 amino acids, and should preferably comprise a high percentage ofglycine residues, preferably at least 50%. Examples of suitable peptidelinkers include peptides (G₄S)₃ (SEQ ID NO:8), G₄IAPSMVG₄S (SEQ IDNO:9), G₄KVEGAG₅S (SEQ ID NO:10), G₄SMKSHDG₄S (SEQ ID NO:11),G₄NLITIVG₄S (SEQ ID NO:12), G₄VVPSLG₄S(SEQ ID NO:13) and G₂EKSIPG₄S(SEQID NO:14). When the scFv is produced in the VL to VH orientation(V_(L)-linker-V_(H)), the distance between the C-terminus of VL andN-terminus of VH is slightly greater than between the C-terminus of VHand N-terminus of VL (39-43 Å versus 30-34 Å). Therefore, a 18 aminoacid residues linker is the minimal sequence size that can be used. Asuitable peptide linker should then have between 18-25 aa, preferablybetween 18-21 aa. An example of a suitable linker is linker of sequenceGSTSGSGKSSEGSGSTKG (SEQ ID NO: 15).

By a “peptide tag” is meant a peptide of 5-15 amino acids for whichspecific antibodies are available. Although optional in a scFv fragmentaccording to the invention, such a peptide tag inserted at theC-terminal end of the scFv fragment permits to facilitate purificationof the scFv fragment after recombinant production. Indeed, the peptidetag gives the protein a specific binding affinity it would nototherwise, which permits an easier purification using chromatography.Examples of suitable peptide tags include a His6 tag (HHHHHH, SEQ IDNO:16), which has affinity towards nickel ions and can thus be purifiedusing a nickel ions containing chromatography column, or epitopespeptides with high affinity for their specific antibody and can thus bepurified using a column containing immobilized antibody directed againstpeptide, such as a c-myc tag (EQKLISEEDLN, SEQ ID NO:17), a HA tag(YPYDVPDYA, SEQ ID NO:18), a Flag tag (DYKDDDDK, SEQ ID NO:19) a proteinC tag (EDQVDPRLIDGK, SEQ ID NO:20), a Tag-100 tag (EETARFQPGYRS, SEQ IDNO:21), a V5 epitope tag (GKPIPNPLLGLDST, SEQ ID NO:22), a VSV-G tag(YTDIEMNRLGK, SEQ ID NO:23) or a Xpress tag (DLYDDDDK, SEQ ID NO:24).

By a “short peptide spacer” is meant a peptide of 1-15 amino acids,preferably 1-12 or 1-10 amino acids, for instance 8 amino acids. Such apeptide spacer is intended to separate the true scFv part (VH and VLdomains separated by a peptide linker) of the optional peptide tag. Itis not necessary in a scFv fragment according to the invention, inparticular when there is no peptide tag, but also when a peptide tag isincluded. Indeed a peptide tag may be directly fused to the VH or VLdomain. However, a short peptide spacer of 1-12 amino acids may beuseful. For instance, in the 9O12.2 scFv fragment, a 8 amino acidspeptide spacer of sequence RSRVTVSS (SEG ID NO: 25) has been used.

In a preferred embodiment, the invention concerns a single chainvariable fragment (scFv) directed against human glycoprotein VI,comprising or consisting of an amino acid sequence having at least 95%,at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%,at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, identitywith SEQ ID NO:1, in which amino acids 26 to 35, 50 to 66, 99 to 109,159 to 174, 190 to 196, and 229 to 237 of said 266 amino acids sequenceare constituted of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, and SEQ ID NO:7 respectively. Such functional variants ofscFv 9O12.2 not only keep identical CDR regions but also have a highidentity percentage with the amino acid sequence of scFv 9O12.2. In aparticular embodiment, the invention concerns a single chain variablefragment comprising or consisting of SEQ ID NO:1.

However, framework regions may be mutated to generate a chimeric, inparticular humanized, scFv fragment directed against human glycoproteinVI. With regards to a reference antibody or antibody fragment of a givenspecies, a corresponding “chimeric” antibody or antibody fragment inwhich framework regions have been replaced by corresponding frameworkregions of another species. More particularly, a “humanized” antibody orantibody fragment is an antibody or antibody fragment with identical CDRregions in which framework regions have been replaced by human frameworkregions.

The scFv 9O12.2 fragment has been derived by the inventors from murinemonoclonal antibody 9O12.2, and thus has mouse framework regions.However, to reduce immunogenic effect of injection of this fragment inhuman patients, a humanized scFv fragment may be desirable, in whichmurine framework regions have been replaced by human framework regions.

Several possible methods of humanizing antibody V-domains have beensuggested. They include CDR-grafting, resurfacing V-domains orpredictive computational analysis that explores a diversity ofsubstitutions in a given V chain sequence with the aim of reducingimmunogenicity whilst maintaining the antigen-specificity and affinityof the original molecule (17-19). None of these methods is simple, andall often result in impaired specificity and/or affinity (20-23).

Despite these difficulties, the inventors have generated a firsthumanized scFv fragment in which the V_(H) and V_(L) domains of murine9012.2 scFv fragment, corresponding to amino acids number 1-120 and136-266 of SEQ ID NO:1, have been respectively replaced by humanizedV_(H) and V_(L) domains SEQ ID NO:26 and SEQ ID NO:27, resulting in ahumanized scFv fragment of amino acid sequence SEQ ID NO:28 (hscFv9O12.2 (1), see FIG. 14).

In a first preferred embodiment, the invention thus concerns a singlechain variable fragment according to any of claims 1-3, in whichframework regions of the V_(H) and V_(L) domains have been replaced byV_(H) and V_(L) domains having framework regions of a human antibody. Inparticular, said VH and VL domains may have been replaced respectivelyby SEQ ID NO:26 and SEQ ID NO:27. More precisely, when an scFv fragmentcomprising or consisting of SEQ ID NO:1 is humanized, it may result in afirst humanized scFv fragment comprising or consisting of SEQ ID NO:28(hscFv 9O12.2 (1), see FIG. 14).

To generate such a humanized scFv fragment, a well known technologycalled CDR grafting may be used, which involves selecting thecomplementary determining regions (CDRs) from a donor scFv fragment, andgrafting them onto a human scFv fragment framework of known threedimensional structure (see, e.g., WO98/45322 (24); WO 87/02671 (25);U.S. Pat. No. 5,859,205 (26); U.S. Pat. No. 5,585,089 (27); U.S. Pat.No. 4,816,567 (28); EP0173494 (29); and references 20-21 and 30-31) orperforming database searches to identify potential candidates. In atypical method aided by computer modeling and comparison to humangermline sequences, the antigen binding loops of the monoclonal antibodyto be humanized are superimposed onto the best fitting frameworks. Theapproach to humanize the scFv 9O12.2 has been adapted from CDR graftingtechnology (32). The CDRs of murine 9O12.2 antibody have been grafted ona human variable-chain framework. The choice of the human antibody hasbeen done using the following criteria: the crystallographic structurehas been elucidated (1VGE in pdb library); the variable domains of thehuman antibody 1VGE present a high degree of sequence homology with thevariable domains of 9O12.2 (VH 60,6%; VL 55,4%); Structure comparison ofthe crystallographic data of the candidate human antibody 1VGE and themodel of the variable domains of 9O12.2 was done and allowed us tovalidate the choice of the human acceptor scaffold 1 VGE. This strategyminimizes the risk of lowering the stability of the interaction betweenVH and VL domains while preserving the scaffold required for correctfolding of the CDR, preserving a high affinity for the antigen. Thehuman antibody 1VGE selected based on these analysis allowed toconstruct the humanized 9O12.2 scFv. For structural raisons, 10 residuesafter CDR H2 were not mutated. The nucleotide sequence encoding thehumanized scFv 9O12.2 was optimized. First, restriction sites betweenCDR regions were introduced to make possible adjustments for optimalbinding characteristics. Optimization was then performed using thebacterial codon usage in order to express the humanized scFv in theprocaryotic expression system E. coli. Said method resulted in ahumanized 9012.2 scFv fragment in which V_(H) and V_(L) domains of themurine 9012.2 scFv fragment, corresponding to amino acids number 1-120and 136-266 of SEQ ID NO:1 respectively, have been replaced by SEQ IDNO:26 and SEQ ID NO:27 respectively, i.e. a humanized 9012.2 scFvfragment consisting of SEQ ID NO: 28. In a preferred embodiment of ahumanized scFv fragment according to the invention, said scFv fragmentthus comprises or consists of SEQ ID NO:28.

Despite promising results obtained with this first humanized scFvfragment, this fragment was not optimal for production in bacteria, anda further optimized second humanized scFv fragment (hscFv 9O12.2(2)) wasderived from hscFv 9O12.2(1). Briefly, for VL FR1 and FR2 regions, theidentity with 1VGE frameworks 1 and 2 were low and the original 9O12 VLFR1 and FR2 were thus preserved in the second humanized scFvconstruction. Other refinements were carried out on the basis of closeinspection of the model (see Example 2 for more details).

Thus, in a second preferred embodiment, the invention thus concerns asingle chain variable fragment according to any of claims 1-3, in whichframework regions of the V_(H) and V_(L) domains have been replaced byV_(H) and V_(L) domains having framework regions of a human antibody. Inparticular, said VH and VL domains may have been replaced respectivelyby SEQ ID NO: 26 and SEQ ID NO: 46. More precisely, when an scFvfragment comprising or consisting of SEQ ID NO: 1 is humanized, it maythus also preferably result in a second humanized scFv fragmentcomprising or consisting of SEQ ID NO: 47 (hscFv 9O12.2 (2), see FIG.18).

The invention further concerns a nucleic acid sequence encoding a singlechain variable fragment according to the invention as described before.In a particular embodiment, when said scFv fragment comprises orconsists of SEQ ID NO: 1, said nucleic acid sequence may comprise orconsist of SEQ ID NO:29, or any derived nucleic sequence encoding SEQ IDNO:1, for instance as a result of the degeneracy of the genetic code.

When said scFv fragment is humanized and V_(H) and V_(L) domains of themurine 9012.2 scFv fragment, corresponding to amino acids number 1-120and 136-266 of SEQ ID NO:1 respectively, have been replaced by SEQ IDNO:26 and SEQ ID NO:27 respectively, resulting in a first humanized9012.2 scFv fragment (hscFv 9O12.2(1)) comprising or consisting of SEQID NO:28, said nucleic acid may comprise or consist of SEQ ID NO:30, orany derived nucleic sequence encoding SEQ ID NO:28, for instance as aresult of the degeneracy of the genetic code.

Alternatively, when said scFv fragment is humanized and V_(H) and V_(L)domains of the murine 9012.2 scFv fragment, corresponding to amino acidsnumber 1-120 and 136-266 47 respectively, resulting in a secondhumanized 9012.2 scFv fragment (hscFv 9O12.2(2)) comprising orconsisting of SEQ ID NO:47, said nucleic acid may comprise or consist ofSEQ ID NO:50 (see FIG. 18C), or any derived nucleic sequence encodingSEQ ID NO:47, for instance as a result of the degeneracy of the geneticcode.

The invention also concerns an expression vector comprising a nucleicacid sequence as described. Such an expression vector also comprisesappropriate nucleic acid sequences necessary for the expression of theoperably linked scFv coding sequence in a particular host cell. Nucleicacid sequences necessary for expression in prokaryotes include apromoter, optionally an operator sequence, a ribosome binding site andpossibly other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals in ahost cell.

Such an expression vector may also contain a leader sequence directingthe expressed scFv to a particular cellular compartment, for instance asequence directing a membrane expression or a secretion of the scFvfragment, or directing its expression in periplasm of bacteria. It mayalso contain under control of the same promoter a selection gene, theexpression of which may be easily detected to select recombinant hostcells transformed with the expression vector. Suitable selection genesinclude notably antibiotics resistance genes, fluorescent genes, or anyother gene which expression may be easily monitored known by a personskilled in the art.

The invention further concerns a host cell comprising an expressionvector as described above. Such a recombinant host cell may be obtainedby transfecting or transforming a host cell with an expression vectoraccording to the invention.

Such a host cell may be either prokaryotic or eukaryotic. Suitableprokaryotic host cells include gram-positive and gram-negative bacteria.Among gram-negative bacteria, a preferred host cell is represented by E.coli. For use in bacteria, the expression vector may preferably containa leader sequence directing the expression of the scFv fragment intobacterial periplasm, corresponding to the space between the plasmamembrane and the outer membrane of gram-negative bacteria or between theplasma membrane and the peptidoglycan layer (cell wall) of gram-positivebacteria. Suitable sequences directing the expression of a polypeptideto bacteria periplasm include ompA, ompF, ompT, LamB, β-lactamase, cpVIII from M13, pelB, malE or phoA signal peptides or leader sequences.In a particular embodiment, said leader sequence is pelB.

Alternatively, a eukaryotic cell may be used, in particular a mammaliancell. Indeed, this may permit to directly generate a glycosylated scFvfragment. Mammalian cell lines available as hosts for expression areknown in the art and include many immortalized cell lines availablenotably from the American Type Culture Collection (ATCC), including butnot limited to, Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. As eukaryotic cells, yeast cells may also be used. To obtaina glycosylation pattern closest to a natural human glycosylationpattern, it may be resorted to human cell lines as host cells.Alternatively, Pichia pastoris yeast cells, a robust organism commonlyused in fermentation processes which can be grown to high cell densityin a chemically defined growth medium, have been modified by firsteliminating endogenous yeast glycosylation pathways, while sequentiallyengineering into the organism a synthetic in vivo glycosylation pathwaythat enables the yeast to produce a complex human N-glycan,GlcNAc2Man3GlcNAc2, in vivo (see EP1297172 (33), EP1522590 (34), andreferences 35, 36). Such modified yeast cells with a humanizedglycosylation pathway are able to secrete a human glycoprotein withuniform complex N-glycosylation.

The invention also concerns a method for preparing a single chainvariable fragment according to the invention as described above,comprising:

-   -   a) culturing a host cell according to the invention as described        above, and    -   b) purifying said single chain variable fragment.

Protocols and media for culturing host cells represent routineprocedures and are readily available to any person skilled in the art.Concerning purification of the obtained scFv fragment, it may beperformed using well-known technologies, including affinitychromatography (e.g., using protein L-Sepharose) or ion-exchangechromatography, hydroxylapatite chromatography, gel electrophoresis,dialysis, etc. More precisely, when the scFv fragment comprises a His6tag or an epitope tag as described above, HPLC with nickel ion columnsor with columns containing immobilized specific antibodies.

The invention further concerns a single chain variable fragmentaccording to the invention as described above, as a medicament.

The invention also relates to a pharmaceutical composition, comprising asingle chain variable fragment according to the invention as describedabove and a pharmaceutically acceptable carrier.

More precisely, the invention also concerns the use of a single chainvariable fragment according to the invention as described above forpreparing a medicament for treating and/or preventing a cardiovasculardisease selected from arterial and venous thrombosis, restenosis, acutecoronary syndrome, and cerebrovascular accidents due to atheroscleroris.In a preferred embodiment of such a use, said cardiovascular disease isthrombosis.

Having generally described this invention, a further understanding ofcharacteristics and advantages of the invention can be obtained byreference to certain specific examples and figures which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

DESCRIPTION OF FIGURES

FIG. 1. Schematic description of the method used to construct scFv9O12.2 expression vector. Step 1: PCR amplification 9O12.2.2 monoclonalantibody VH segment using T7 and Link9O12.2VHFor primers and VL segmentusing Link9O12.2VLRev and 9O12.2mycFor primers. Step 2: Overlap PCRamplification using pSWRev and 9O12.2mycFor primers followed by PCRamplification using pSWRev and pSWFor primers, thus generating a nucleicacid coding scFv 9O12.2. Step 3: DNA purification of PCR products. Step4: Digestion of purified DNA using Pst1 and Xho1 restriction enzymes.Step 5: Cloning of the digested product into pSW1 expression vector.Step 6: Sequencing of the obtained construction.

FIG. 2. Nucleotide SEQ ID NO:50 and deduced amino-acid (SEQ ID NO:1)sequences of scFv 9O12.2 cloned into plasmid pSWscFv9O12.2myc.Nucleotide and deduced amino-acid sequences of scFv 9O12.2 loned betweenthe restriction sites Pst1 and Xho1 into the expression vector pSW1. Thenucleotide sequences corresponding to the primers used for PCRamplification or encoding the linker peptide are shown in italics. Thededuced amino-acid sequence of the complementary determining regions(CDRs) of VH and VL are underlined. The deduced amino-acid sequence ofthe c-myc flag is shown.

FIG. 3. Three-dimensional model of the scFv9O12.2. Model of the 9O12.2.2antigen-binding domains (side view) The CDRs are indicated.

FIG. 4. Analysis of bacterial periplasmic extracts by flow cytometry onhuman platelets. Bacterial periplasmic extracts of induced recombinantbacteria transformed with a plasmid encoding an irrelevant scFv-myc orthe plasmid pSW scFv9O12.2-myc were incubated with human platelets.Binding of scFvs was analysed using an FITC-coupled anti-cmyc antibody.A: irrelevant scFv, B: scFv 9O12.2. Left panel: forward versus sidescatter; right panel: fluorescence histogram.

FIG. 5. Periplasmic production and affinity-chromatography purificationof a recombinant protein from pSW1-scFv9O12.2myc cultures in E. coliTopp1. (A): SDS-PAGE stained with Coomassie Brillant Blue. (B): Westernblot analysis with anti-c-myc IgG. Lane 1, Molecular mass standards;lane 2, periplasmic fraction of induced bacteria loaded onto aGPVI-sepharose column; lane 3, GPVI-sepharose column flow throughfraction; lane 4 GPVI-sepharose column eluted fraction.

FIG. 6. Size exclusion chromatography of native scFv. The size exclusionchromatography of affinity purified scFv 9O12.2 was performed on aSuperdex 75 HR 10/30 column calibrated with standards of known molecularmass.

FIG. 7. Binding of purified scFv 9O12.2 to immobilized GPVI-Fc. scFv9O12.2 (dark) or irrelevant scFv (grey symbol) was incubated on GPVI-Fccoated plates and detected using HRP coupled anti-c-myc (A): bindingisotherm. (B): scFv 9O12.2 (60 μg.mL⁻¹) binding in competition withincreasing amounts of IgG 9O12.2.

FIG. 8. Surface Plasmon Resonance analysis of scFv 9O12.2 binding toimmobilized GPVI-Fc. scFv 9O12.2 affinity for GPVI-Fc is high with aK_(D) in the order of 2.5×10⁻⁹ M. K_(D) of IgG and Fab 9O12.2.2 forGPVI-Fc are also in the nanomolar order (6.5×10⁻⁹ M and 4.5×10⁻⁹ Mrespectively).

FIG. 9. Inhibition of GPVI binding to immobilized collagen. GPVI-Fc wasincubated on immobilized collagen in the presence of increasing amountsof antibodies: scFv 9O12.2 (triangle), or pFab (square) (A): 2 μg of GPVI-Fc; (B): 4 μg of GPVI-Fc Bound GPVI-Fc was detected using an HRPcoupled anti-Fc IgG.

FIG. 10. Binding of purified scFv 9O12.2 to platelets: Flow cytometryanalysis. Washed platelets were incubated with an irrelevant scFv (top)or decreasing amounts of scFv 9O12.2 (100, 50 and 25 mM). Binding ofscFvs was analysed using an FITC-coupled anti-c-myc IgG.

FIG. 11. Effect of scFv 9O12.2 on platelet aggregation induced bycollagen. Washed human platelets (2.10⁸ mL) were incubated with anirrelevant scFv (bottom curve), Fab 9O12.2 (25 μg/ml) (upper curve),isolated monomeric scFv 9O12.2 (black) for 5 min at 37° C. and thecollagen was added. Aggregation was analyzed at 37° C. with stirringconditions and change in light transmission was recorded.

FIG. 12. Effect of _(m)scFv 9O12 on platelet aggregation induced bycollagen under arterial flow conditions. Whole blood (5 mL) was labeledwith the cell permeable fluorochrome DIOC-6 platelets appear in white onthe picture was incubated with PBS (A) or antibody fragments at 50μg.mL⁻¹ (B-D), and then perfused onto collagen-coated coverslips in aflow-chamber at 1 500 s′. The formation of platelet aggregates bound tothe collagen matrix was recorded with a fluorescent microscope. (B)irrelevant scFv 9C2. (C) mscFv9O12. (D) Fab 9O12.

FIG. 13 Effect of mscFv 9O12 on thrombin generation induced by collagenin PRP. Platelet rich plasma (PRP) was preincubated with vehicle (blackcurve) or 50 μg.mL⁻¹ of Fab9O12 (dark gray) or _(m)scFv 9O12 (lightgray) before adding collagen (5 μg.mL⁻¹). Thrombin generation wasinitiated by adding 0.5 pM tissue factor and 16.6 mM CaCl₂. Thrombinconcentration was determined using a fluorescent substrate and wascalculated relative to a calibrator. The traces are from onerepresentative experiment. Bars graphs represent mean±SD of the lagphase and the peak values (n=3) (The bars corresponding to the SD aretoo small to be visible).

FIG. 14. Nucleotide sequence and deduced amino acid sequence ofhumanized VH and VL domains and of the first humanized 9O12.2 scFvfragment (hscFv 9O12.2(1)). A. Humanized 9O12.2 VII (1) nucleotides (SEQID NO: 31) and amino acids (SEQ ID NO:26) are displayed. B. Humanized9O12.2 VL (1) nucleotides (SEQ ID NO: 32) and amino acids (SEQ ID NO:27)are displayed. C. Humanized hscFv9O12.2(1) nucleotides (SEQ ID NO:30)and amino acids (SEQ ID NO:28) are displayed.

FIG. 15. Characterization of the first humanized scFv 9O12.2(hscFv9O12.2(1)). Affinity binding to GPVI sepharose: The periplasmic fractionof recombinant bacteria was loaded onto a GPVI sepharose column.Retained proteins were analyzed by Western-Blot using an anti-c-myc IgGfollowed by anti-mouse antibody coupled to HRP and ECL revelation. Asingle band is detected with a molecular mass of 28.5 kDa as expectedfor the humanized scFv.

FIG. 16. Binding of scFv 9O12.2 to GPVI-Fc. Purified first humanizedscFv (hscFv 9O12/(1), dark grey) and mscFv (light grey) were injected onGPVI-Fc immobilized on a CM5 sensorchip. Sensorgrams are shown afterdeduction of the blank signal.

FIG. 17. Binding of the first humanized scFv9O12 (hscFv 9O12.2 (1)) tohuman platelets. Human platelets were incubated with bacterialperiplasmic extracts. scFv bound to platelets was detected using FITCcoupled anti-c-myc antibody. Binding to platelet was analyzed on XLEpics Coulter Flow cytometer. An irrelevant scFv was used as a negativecontrol (upper panel). The shift to the right of the histogram (lowerpanel) indicates that the humanized scFv 9O12.2 binds to platelet GPVI.

FIG. 18. Nucleotide sequence and deduced amino acid sequence ofhumanized VII and VL domains and of the second optimized humanized9O12.2 scFv fragment (hscFv 9O12.2(2)). A. Humanized 9O12.2 VH (2)nucleotides (SEQ ID NO: 48) and amino acids (SEQ ID NO:26) aredisplayed. B. Humanized 9O12.2 VL (2) nucleotides (SEQ ID NO: 49) andamino acids (SEQ ID NO:46) are displayed. C. Humanized hscFv9O12.2(2))nucleotides (SEQ ID NO:50) and amino acids (SEQ ID NO:47) are displayed.Enzymes restriction sites are in bold. The linker between VH and VLdomains is underlined. CDR regions are highlighted in grey.

FIG. 19. Optimized humanization of the 9O12 antibody variable domains.Sequence analysis of antibody V-domains: murine 9O12 (m9O12; VH is SEQID NO:26, VL is SEQ ID NO:27); 1VGE (VH is SEQ ID N0:52, VL is SEQ IDNO:53): humanized 9O12 (h9O12; VH is AA 1-120 of SEQ ID NO:47, VL is AA136-247 of SEQ ID NO:47)) and 1×9Q (VL is SEQ ID NO: 51). (.) indicatesresidues identical to murine 9O12. (-) indicates a gap. Residues of thehumanized V-domains having no similarity with murine 9O12 are shown inred and blue (residues A_(H71), K_(H73), R_(H76), L_(L59), D_(L60)according to the Kabbat nomenclature). CDRs are highlighted in gray.

FIG. 20. Characterization of the second humanized (hscFv 9O12.2 (2).A—Western-blot detection of recombinant scFvs using the anti-cMycantibody. B—Flow cytometry analysis. Human platelets were pre-incubatedwith antibody fragments for 30 minutes. scFvs binding was detected usingFITC conjugated anti-c-Myc antibody

EXAMPLES Example 1 Synthesis and Activity of a Murine and a FirstHumanized Single Chain Variable Fragment scFv 9O12.2 Directed AgainstHuman Glycoprotein VI

1.1 Experimental Procedure

1.1.1 Material

Media and Solutions

LB (Luria-Bertani), DIFCO 402-17; LB-Agar, DIFCO 445-17; 2×TY(Trypton-Yeast), DIFCO 244020; TES: Tris-HCl 30 mM, EDTA 1 mM, sucrose20%, pH 8.5; PBS: NaCl 0,14 M, KCl 13 mM, KH₂PO₄ 9 mM, Na₂HPO₄ 50 mM, pH7.4; BBS: boric acid 50 mM, NaCl 150 mM, pH 7.6; Ampicillin, EuromedexEU-0400C; Isopropyl β-D-thiogalactoside (IPTG), Euromedex EU0008-B;Bovine serum albumin (BSA), Sigma B4287; FITC-coupled anti-c-myc IgG,Sigma F-2047; desoxyribonuclease A, aprotinin: Sigma A-6279; HRP-coupledanti-c-myc IgG, Invitrogen R951-25; orthophenyl diamine (OPD): SigmaP8787, Trizol: Invitrogen, Collagen Horm type I: Nycomed, pGEM_(T)cloning vector, Promega. The pSW1 plasmid was used for scFv expression.6H8 and 9C2 was used as non relevant scFv.

Oligodesoxyribonucleotides Sequences

The used oligodesoxyribonucleotides are listed in the following Table 2:

TABLE 2 Oligodesoxyribonucleotides Name 5′-3′ sequence SEQ ID NO T7TAATACGACTCACTAGGGCGAAT SEQ ID NO: 33 pSWRev CGGCAGCCGCTGGATTGTTASEQ ID NO: 34 pSWFor CGAGCTTAGCCCTTATAATTCAG SEQ ID NO: 35 ATCCTCLink901- GGAGGCGGATCCGGTGGTGGCGG SEQ ID NO: 36 2VLRevATCTGGAGGTGGCGGAAGCGATG TTTTGATGACCCAAACTCCACT Link901-ACCACCGGATCCGCCTCCGCCTGA SEQ ID NO: 37 2VHFor GGAGACGGTGACCGT 9O12.2-GACCCTCGAGCGTTTGATCTCCAG SEQ ID NO: 38 mycFor CTTGGT 9O12.2-GGCGACTGGTACTTCGATGTC SEQ ID NO: 39 H3Rev 9O12.2-AAATGGTGACACTTCCTTCAATCA SEQ ID NO: 40 H2Rev 9O12.2-GTTTTCAAGGCTCTGACTAGACCT SEQ ID NO: 41 L1For MKC5ForGGATTACAGTTGGTGCAGCATC SEQ ID NO: 42 MKRevU GAYATTGTGMTSACMCARWCTSEQ ID NO: 43 VHFor CGGGATCCTCTAGACAGTGGATAR SEQ ID NO: 44 ACMGATGGVHRev CGGGATCCTCTAGAGGTSMARCT SEQ ID NO: 45 GCAGSAGTCWGGPreparation of Human Platelets

Human platelets were prepared as previously described (9)

Flow Cytometry

“Simple FITC labelling of platelets program” flow cytometer CoulterEpics XL was used

Preparation of GPVI-Sepharose:

Reagents: CnBr-sepharose was from Amersham-Pharmacia. Recombinantsoluble GPVI (GPVI-Fc) was produced and purified in the laboratory (9).

Method: CnBr-sepharose (Amersham-Pharmacia) was prepared as indicated bythe supplier. It was incubated with GPVI-Fc (8 mg/mL of gel in NaHCO₃0.1 M; NaCl 0.5 M pH 8.3) for 18H under agitation. The gel was filteredand the protein concentration in the filtrate was measured to determinethe yield of the coupling. After a blocking step with ethanolamine (1 M,pH 8.8) for two hours at room temperature in the dark with gentlestirring, the gel was filtered and washed successively with the couplingbuffer and with sodium acetate 0.1 M, NaCl 0.5 M pH 4. The gel wasstored at 4° C. in PBS containing sodium azide (1%).

1.1.2 Procedures

Genetic Construction of scFv 9O12.2:

mRNA was isolated from freshly subcloned hybridoma 9O12.2.2 cells whichproduce the immunoglobulin G (IgG) 9O12.2.2 directed against human GPVI.cDNA encoding the antibody variable domains were cloned after RT-PCR andthe scFv 9O12.2 gene was created by PCR splicing with overlapextensions. An expression vector derived from pUC 19 [pSW I vector (37)]was used for the production of the scFv 9O12. It contains the LacZpromotor inducible with IPTG, the pelB leader sequence and, downstream,the gene coding for the scFv 9O12.2 fused to the flag c-myc and a geneof resistance to ampicillin used to select the recombinant bacteria.

The method used to construct the scFv 9O12.2 expression vector issummarized in FIG. 1.

Production of the scFv 9O12.2

The leader sequence pelB allows the recombinant scFv 9O12.2 to beaddressed to the periplasm of bacteria transformed with the expressionvector pSW scFv 9O12.2myc. The bacteria Toppl® (non K12, Rif^(r), F′,proAB, lacI^(q)ZΔM15, Tn10, tet^(r)) (Stratagene, La Jolla, USA), colony55T1 is used to produce the scFv 9O12.2.

-   -   J0: Plating of the 55T1 colony on LB-Agar supplemented with        ampicillin (sub-cloning). Incubation overnight at 37° C.    -   J1: 4 PM: Selection of one clone and culture in 5 mL LB        supplemented with ampicillin. Incubation overnight at 37° C.        with agitation (125 rpm).    -   J2: 8 AM: Measurement of the absorbance at 600 nm (expected        value A_(600nm)=1.5±0.1). Transfer of 4 mL to 500 mL        2×TY+Ampicillin. Incubation at 37° C. with agitation (125 rpm)        until A_(600nm) reaches 1.5±0.1 (˜8 H). Then, induction of the        bacteria with 0.8 mM IPTG. Incubation for 16 H at 16° C. with        agitation (75 rpm).    -   J3: periplasmic proteins extraction: Bacterial cells are        collected by centrifugation (3600 g, 20 min at 4° C.). Pellet is        gently resuspended in 10 mL TES buffer and incubated for 30 min        on ice. The cells are then subjected to a mild osmotic shock by        addition of TES buffer, diluted 1:4. After incubation on ice for        30 min., insoluble material is removed by centrifugation at 15        000 g for 30 min at 4° C. Then deoxyribonuclease A (50 U) and        proteases inhibitor (Aprotinin 2 μg/mL) are added to the        supernatant correponding to the periplasmic proteins extract.        After that, the preparation is extensively dialysed against PBS        at 4° C. A periplasmic proteins extract of bacteria expressing        an irrelevant scFv is prepared according to the same procedure.    -   J4: Centrifugation at 15 000 g for 30 min at 4° C. The        supernatant is collected and its absorbance at 280 nm measured        (expected value: 1.5 to 3.0). One sample is taken for analysis        and the preparation (˜35 mL±2.0) is stored at −20° C.    -   J5: Screening of the periplasm extracts by flow cytometry:        washed human platelets (2×10⁷/mL) are incubated with 100 μL of        periplasmic proteins extract for 30 min at room temperature. The        FITC-coupled anti-c-myc IgG is added and the incubation        continued for 30 min at room temperature in the dark. A negative        control is performed with the anti-c-myc antibody in the absence        of periplasm extracts. Samples are analysed by flow cytometry        (Coulter Epics XL).    -   J6: Purification of the scFv 9O12.2 by affinity chromatography        on GPVI-coupled sepharose. The periplasm extract (35 mL) is        incubated with 500 μL GPVI-Sepharose for 12H at 4° C. and 4H at        room temperature. The mixture is loaded on a column. The        flow-through fraction is collected before washing with PBS until        A_(280nm)=0.001; Elution is performed with Glycine 0.1 M pH 3.0        and fractions of 400 μL are collected in tubes containing 5 μL        Tris 3 M on ice. A_(280nm) is measured and fractions with        A_(280nm) higher than 0.2 are pooled and extensively dialysed        against PBS.    -   J7: The sample is centrifuged at 15000 g for 30 min. Protein        concentration is determined after measuring A_(280nm). The        ProtParam software is used to determine the theorical Mr of the        scFv9O12.2myc and its extinction coefficient at 280 nm.        Protein Analysis:

Protein analysis was made by electrophoresis in polyacrylamide slab gelsin the presence of SDS according to Laemmli; by immunoblotting aftertransfer of the proteins to nitrocellulose, incubation with an HRPcoupled anti-c-myc antibody and detection using 4 chloro-naphtol.

Experimental Determination of the Mass

Mr of purified scFv is determined on a matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometer(Voyager DE-PRO; PerSeptive Biosystems Inc., Framingham, Mass.). Thesaturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrileand 0.1% TFA is used as matrix and the spectrometer is calibrated usingexternal standard peptide mixtures.

Size Exclusion-Diffusion Chromatography:

Purified scFv preparation were resolved by size-exclusion diffusionchromatography on a Superdex 75 HR column calibrated with standards ofknown molecular mass. 200 μL of sample was injected with a rate of 0.5mL/min. Detection is monitored using UV detector at 226 nm. Fractionswere used immediately for analysis.

Binding to Soluble Recombinant GPVI

Microtiter plates are coated with GPVI-Fc (10 μg/mL, 100 μL per well)overnight at 4° C. Wells are saturated with 100 μL BSA for 2 hours atroom temperature. Then, increasing concentrations of scFv 9O12 are addedto the wells for 2 hours. Bound scFvs are detected after incubation for2 h at room temperature with HRP-coupled anti-c-myc-antibody (1/750 enPBS). Then, the substrate solution (OPD) is added to the wells for 5min. and absorbance is read at 492 nm. Two controls are performed: thefirst one using an irrelevant scFv in place of scFv 9O12.2 and thesecond one by omitting coating with GPVI-Fc. Five washes with PBScontaining 0.05% Tween and 0.1 mg/mL BSA are carried out between eachintermediate step.

Construction of the First Humanized scFv Fragment (hscFv 9O12.2(1))

The approach to humanize the scFv 9O12.2 has been adapted from CDRgrafting technology 9. The CDRs of murine 9O12.2 antibody have beengrafted on a human variable-chain framework. The choice of the humanantibody has been done using the following criteria: thecrystallographic structure has been elucidated (1 VGE in pdb library);the variable domains of the human antibody 1VGE present a high degree ofsequence homology with the variable domains of 9O12.2 (VH 60,6%; VL55,4%); Structure comparison of the crystallographic data of thecandidate human antibody 1 VGE and the model of the variable domains of9O12.2 was done and allowed us to validate the choice of the humanacceptor scaffold 1VGE. This strategy minimizes the risk of lowering thestability of the interaction between VH and VL domains while preservingthe scaffold required for correct folding of the CDR, preserving a highaffinity for the antigen. The human antibody 1VGE selected based onthese analysis allowed us to construct the humanized 9O12.2 scFv. Thenucleotide sequence encoding the humanized scFv 9O12.2 was optimized.First, restriction sites between CDR regions were introduced to makepossible adjustments for optimal binding characteristics. Optimizationwas then performed using the bacterial codon usage in order to expressthe humanized scFv in the procaryotic expression system E. coli.

More precisely, we first constructed a 3D-structural model of_(m)scFv9O12 in silico after identifying the crystal structures withsequences most similar to the 9O12 variable domains. All these sequenceswere of murine origin. The top four scoring structures of murine originwere used for modeling. For the VH gene, we used 1PLG, 1MNU, 1A5F and MIwhich have 66-78% sequence identity (79-85% similarity) with 9O12. Forthe VL gene, we used 1PLG, 1IGI, 1MNU and 1AXT, which have 87-90%sequence identity (94-95% similarity). The 3D structures of all thesesequences were solved with a resolution higher than 2.8 Å. Twenty modelswere generated for each domain using Modeler 3.0 software, and the bestone was selected on the basis of the RMSD value (0.13 Å for VH and 0.703Å for VL) and detailed inspection.

We then proceeded to the humanization of 9O12 V-domains. To do this,FASTA searches were performed to independently align VH and VL aminoacid sequences against a repertoire of human antibody sequencesregistered in the PDB data bank. Among the human V-domains that matched9O12 we first selected a VH and a VL from the same antibody molecule inorder to preserve the interdomain contacts that occur in a naturalantibody. The human antibody 1VGE was selected because it had the bestidentity score with 9O12 when the entire V-domain sequences werespanned, and found to exhibit 62% and 55% identity for VH and VL,respectively. When calculated over framework region sequences alone, theidentity was even slightly better, showing 69.5% and 65.4% identity,respectively. In addition, the crystallographic structure of 1 VGE wassolved at high resolution (2 Å and R value 0.18). We therefore decidedto graft 9O12 CDRs onto the 1 VGE template in silico. A gene encodingthis construct was chemically synthesized and inserted into pSWI exactlyas had been done for _(m)scFv9O12. TOPPI cells transformed with thisvector were induced to express the recombinant protein.

This resulted in a first humanized scFv fragment named hscFv 9O12.2(1)

Competition with 9O12.2 IgG:

The scFv9O12.2 (100 μg/ml) was mixed with increasing concentrations of9O12.2 IgG before addition to GVI-Fc coated microtitration wells. BoundscFv was detected as above.

Effect of the Antibody on GPVI Binding to Collagen:

Microtitration wells are coated with fibrilar type I collagen (CollagenHorn, Nycomed, Munich 2 μg/well), saturated with BSA and washed.Increasing amounts of GPVI-Fc preincubated with PBS, scFv 9O12.2 or Fab9O12.2 are added to the wells. After one hour at room temperature andwashing bound GPVI-Fc is detected using a peroxydase-coupled anti-humanFc and OPD.

Surface Plasmon Resonance (SPR, BIAcore):

Binding of the anti-GPVI scFv to recombinant GPVI-Fc is analyzed withsurface plasmon resonance using a BIAcore 2000 system (Uppsala, Sweden).Binding studies is performed with scFv 9O12, Proteolytic Fab andparental IgG.

Recombinant GPVI-Fc is immobilized (˜600 RU) onto a Carboxy-MethylDextran CM5 sensor chip using the amine coupling method (Wizardprocedure). Antibody is then passed over the immobilized recombinantGPVI-Fc in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005%polysorbate 20 (v/v)) at a flow rate of 20 μl/min at 25° C. Kineticconstants (k_(a), k_(d)) and affinity are determined using BIAevaluationversion 3.1 software, by fitting data to binding model. HBS-EP is therunning buffer. 10 mM glycine-HCl pH 2.5 injected for 30 s at 20 μl/minis the regeneration buffer. All reagents and buffers used are fromBiacore.

Binding of the Purified scFv to Human Platelet GPVI

Washed human platelets (2.10⁷/mL) are incubated for 30 minutes at roomtemperature with 0 to 100 μg/ml of purified scFv 9012 and then incubatedagain for 30 minutes at room temperature in the dark with 5 μL of FITCcoupled anti-c-myc IgG (dilution 1:60). Cell fluorescence is measuredusing a flow cytometer (Epics XL, Coulter). Background is determined byusing an irrelevant scFv in place of scFv 9O12.2.

Platelet Aggregation

Washed human platelets (3×10⁸/mL) are preincubated for 5 min at 37° C.without stirring with PBS, the scFv 9O12.2 or the Fab 9O12.2.Aggregation is initiated by adding type I collagen and the changes inlight transmission is continuously recorded (Chronolog Aggregometer).

Platelet Aggregation Under Flow Conditions

Platelet adhesion to collagen under flow conditions was measuredessentially as described elsewhere (9). Glass coverslips were coatedwith fibrillar type I collagen (50 μg.mL⁻¹). Blood from healthyvolunteers was collected on 40 μM PPACK, and labelled with DIOC-6 (1μM). Blood aliquots were incubated for 15 minutes at room temperaturewith buffer or purified antibody fragment (Fab 9O12, _(m)scFv9O12, scFv9C2) at a final concentration of 50 μg.mL⁻¹. The mixture was thenperfused over the collagen-coated coverslips inserted in a flow-chamberat 1500 s⁻¹ for 5 minutes. Transmission and fluorescent images wererecorded in real time using a fluorescent microscope. Fluorescent imageswere obtained from at least ten different collagen-containingmicroscopic fields which were arbitrary chosen at the end of perfusion.Area coverage of fluorescent images was analyzed off-line using Histolabsoftware (Microvision, Evry, France).

Thrombin Generation

Thrombin generation was continuously measured in platelet rich plasma(PRP) using the thrombogram method as previously described (38).Briefly, citrated PRP (1.5×10⁸ platelets mL⁻¹) was incubated with theantibody fragments for 10 min at 37° before adding the collagen. Tenminutes later, thrombin generation was initiated by transferring thesamples into the wells of a microtitration plate containing tissuefactor (0.5 pM). After 5 min at 37° C., the reaction was initiated byadding buffer containing CaCl₂ and the fluorescent thrombin substrateZ-GGR-AMC (Stago, Asnières, France)). Fluorescence accumulation of thecleaved substrate was continuously measured at excitation and emissionwavelengths of 390 and 460 nm respectively. First derivative curves offluorescence accumulation were converted into thrombin concentrationcurves using a thrombin calibrator (39). The peak height is an indicatorof the maximum rate of thrombin formation, and is sensitive to plateletactivation.

1.2 RESULTS

1.2.1 Murine 9O12.2 scFv (mscFv 9O12.2)

Cloning of Antibody 9O12.2.2 VH and VL cDNAs 9O12.2 is a murine IgG₁(kappa chain). Total RNA was extracted from about 5.10⁸ freshlysubcloned hybridoma cells in a single-step procedure using Trizol. ThisRNA preparation was used as a template for first-strand cDNA synthesis.Double strand cDNAs encoding IgG 9O12.2.2 VH and VL domains were thenamplified by PCR using primers sets (VHRev, VHFor) and (MKRevU, MKC5For)respectively. VH cDNA sequence was unique and this was confirmed bysequencing of the cDNA after cloning into pGEMT. The sequence of the PCRproduct corresponding to the VL domain was scrambled. After cloning intopGEMT and sequencing of several clones, two VL sequences wereidentified. Data analysis using fasta analysis showed that one of the VLsequence derived from the MOPC-21 clone that express an endogeneous andaberrant non-functional Vk mRNA (Gene bank acc number M35669). Thenucleotide and deduced amino acid sequences of the other VL gene and theVH gene are shown in FIG. 2 as part of the scFv 9O12.2. These sequenceswere compared with immunoglobulin variable region sequences registeredin several data banks (UNIPROT UNIREF100 UNIREF90 UNIREF50 UNIPARCSWISSPROT IPI PRINTS SGT PDB IMGTHLAP, PATENT: epop jpop uspop). (DNAbanks: EMBL, HUMAN, MOUSE, SYNTHETIC, PATENT). This allowed us toidentify the three loops corresponding to the complementary determiningregions of the VH and VL regions and the amino-acids residues involvedin the canonical structures.

CDRs were identified according to the following rules as deduced fromKabat et al. (1991) and Chotia and Lesk (1987):

CDR-L1:

Start—Approx residue 24

Residue before is always a Cys

Residue after is always a Trp. Typically TRP-TYR-GLN, but also,TRP-LEU-GLN, TRP-PHE-GLN, TRP-TYR-LEU

Length 10 to 17 residues

CDR-L2:

Start—always 16 residues after the end of L1

Residues before generally ILE-TYR, but also, VAL-TYR, ILE-LYS, ILE-PHE

Length always 7 residues.

CDR-L3:

Start—always 33 residues after end of L2

Residue before is always Cys

Residues after always PHE-GLY-XXX-GLY

Length 7 to 11 residues

CDR-H1:

Start—Approx residue 26 (always 4 after a CYS) [Chothia/AbM definition]Kabat definition starts 5 residues later

Residues before always CYS-XXX-XXX-XXX

Residues after always a TRP. Typically TRP-VAL, but also, TRP-ILE,TRP-ALA

Length 10 to 12 residues (AbM definition) Chothia definition excludesthe last 4 residues

CDR-H2:

Start—always 15 residues after the end of Kabat/AbM definition) ofCDR-H1

Residues before typically LEU-GLU-TRP-ILE-GLY, but a number ofvariations

Residues after LYS/ARG-LEU/ILE/VAL/PHE/THR/ALA-THR/SER/ILE/ALA

Length Kabat definition 16 to 19 residues (AbM definition ends 7residues earlier)

CDR-H3:

Start—always 33 residues after end of CDR-H2 (always 2 after a CYS)

Residues before always CYS-XXX-XXX (typically CYS-ALA-ARG)

Residues after always TRP-GLY-XXX-GLY

Length 3 to 25 residues

The VH cDNA sequence showed 92.96% identity with the cDNA encodinganother murine VH domain (MMMD52C). For the VL we found 98.51% identitywith the cDNAsequence encoding the VL domain of murine anti-acetyl-Lys(BD174891). The deduced amino acid sequences analysis also showed highhomology with the sequence of murine Ig. 9O12.2 VH showed 89.256%identity with the deduced amino-acid sequence of an anti-CD20 murine IgG(BD688655). 9O12.2 VL showed 96.43% identity with the deduced amino-acidsequence of an anti-acetyl Lys murine IgG (BD581288).

Design of the scFv 9O12.2

The nucleotide and deduced amino acid sequences of the scFv 9O12.2 isshown in FIG. 2. The scFv is 266 amino acids long and is composed of theVH and VL sequences linked together by a [G₄S]₃ linker and followed by ashort spacer (8 residues) and the c-myc flag sequence (11 residues).

Theoretical Structural Characteristics of the scFv

The theoretical molecular mass of the scFv is of 28393.5 as determinedusing ProtParam software. Its pI is 6.92 and its extinction coefficient49640 M⁻¹ cm⁻¹ at 280 nm (Table 3).

TABLE 3 Theoretical structural characteristics of scFv 9O12 as deducedfrom bioinformatic analysis using ProtParam software Number of aminoacids: 266 Molecular weight: 28393.5 Theoretical pl: 8.82 Amino acidcomposition: Ala (A) 11 4.1% Arg (R) 9 3.4% Asn (N) 9 3.4% Asp (D) 114.1% Cys (C) 4 1.5% Gln (Q) 15 5.6% Glu (E) 11 4.1% Gly (G) 37 13.9% His(H) 2 0.8% Ile (I) 7 2.6% Leu (L) 24 9.0% Lys (K) 13 4.9% Met (M) 4 1.5%Phe (F) 9 3.4% Pro (P) 8 3.0% Ser (S) 36 13.5% Thr (T) 21 7.9% Trp (W) 62.3% Tyr (Y) 11 4.1% Val (V) 18 6.8% Total number of negatively chargedresidues (Asp + Glu): 22 Total number of positively charged residues(Arg + Lys): 22 Atomic composition: Carbon C 1247 Hydrogen H 1934Nitrogen N 340 Oxygen O 403 Sulfur S 8 Formula: C1247H1934N340O403S8Total number of atoms: 3932 Extinction coefficients: Extinctioncoefficients are in units of M−1 cm−1, at 280 nm. Ext. coefficient 49640Abs 0.1% (−1 g/l) 1.748 assuming ALL Cys residues appear as halfcystines Ext. coefficient 49390 Abs 0.1% (−1 g/l) 1.739 assuming NO Cysresidues appear as half cystines Estimated half-life: The N-terminal ofthe sequence considered is-Q (Gln). The estimated half-life is: 0.8hours (mammalian reticulocytes, in vitro). 10 min (yeast, in vivo) 10hours (Escherichia coli, in vivo) Aliphatic index: 69.21 Grand averageof hydropathicity (GRAVY): −0.333Molecular modelling of scFv 9O12.2

Pdb files of murine variable domains used as a template are:

for the VH domain: 1PLG :H; 1MNU :H; 1A5F :H; 1IGI:H.

for the VL domain: 1PLG :L; 1IGI :L; 1MNU :L; 1AXT :L.

Modeler 3.0. software was used for the modelling of variable domains.Twenty models were designed for each domain and the best was selectedaccording to the RMSD value: VH(RMSD: 0.13 Å); VL (RMSD: 0.703 Å). Themodel is shown in FIG. 3.

Analysis of the Periplasmic Extracts

To assess whether the scFv had the expected anti-GPVI activity, theperiplasmic extracts were tested using human platelets and anFITC-coupled anti-c-myc IgG. FIG. 4 shows a shift to the right of theplatelet fluorescence in the presence of the 9O12.2 scFv (40% positiveplatelets) but not in the presence of the irrelevant scFv (0.17%positive platelets). Furthermore, when the periplasmic extracts wereincubated with immobilised recombinant soluble GPVI only binding of thescFv 9O12.2 was detected. Together, these results indicate that the9O12.2 scFv retains anti-GPVI properties.

Production and Purification of the 9O12.2 scFv

The proteins contained in the periplasmic extract, the flow throughfraction and the fraction eluted from the GPVI-sepharose column wereanalysed by SDS-PAGE and by immunoblot (FIGS. 5A and 5B). The elutedfraction appears as a major band of ˜28 kDa that is in agreement withthe theoretical mass. This band is also detected by immunoblot using ananti-c-myc antibody. These results indicate that affinity chromatographyallows obtaining a rather pure scFv fraction.

Size Exclusion Chromatography

When the purified preparation of scFv was applied on a Superdex 75 HR10/30 column one major peak of ˜25 kD corresponding to monomeric scFvwas observed (FIG. 6). This peak was preceded by two less importantpeaks at 45 and 68 kDa corresponding to dimeric and oligomeric scFv.

The monomeric scFv was very stable, and the multimeric formsspontaneously reverted back to monomers when stored at 4° C., which isessential to preserve monovalent binding to GPVI and biological effects.Indeed, it is well-established that bivalent anti-GPVI molecules (IgGsor F(ab)′₂) can cause platelet activation (40; 93).

Binding of the 9O12.2 scFv to Purified GPVI-Fc and Competition with the9O12.2 IgG

The purified scFv dose dependently bound to purified GPVI-Fc immobilizedon a microtitration plate (FIG. 7A). Specificity of the binding isindicated by the fact that a non relevant scFv did not bind to GPVI inthe same conditions.

Furthermore binding of the scFv to purified GPVI was dose dependentlyinhibited by the parental IgG (FIG. 7B).

Affinity of the 9O12.2 scFv for Soluble GPVI

The affinity of the 9O12.2 IgG and of its Fab prepared after papaindigestion (pFab) for GPVI-Fc determined by surface plasmon resonance arehigh with Kd of respectively 6.5 10⁻⁹M and 4.5 10⁻⁹M. The Kd of the scFvis of 2.5 10⁻⁹M (FIG. 8).

In a second series of experiments, the following values of kineticparameters were determined: k_(on)=6.5×10⁴ M⁻¹s⁻¹, k_(off)=1.7×10⁻¹s⁻and the dissociation constant K_(D)=2.6 nM for the 9O12.2 scFv; andK_(D)=2.3 nM for the 9O12 proteolytic Fab fragment and K_(D)=4.0 nM forthe parental IgG. These values are very similar to those obtained in thefirst experiments and confirm the nanomolar K_(D) value.

Thus, the murine scFv 9O12 still has very high functional affinity, witha K_(D) value in the range of 10⁻⁹M, and this certainly contributes tothe stability of the scFv-platelet GPVI complexes, which is essential toinhibit platelet aggregation under flow conditions.

The Purified 9O12.2 scFv Inhibits GPVI Binding to Collagen.

The 9O12.2 scFv dose dependently inhibited the binding of purifiedGPVI-Fc to immobilized fibrillar collagen type I. (FIG. 9). Itsinhibitory capacity is close to that of the 9O12.2 pFab.

More precisely, mscFv 9O12 inhibited GPVI binding to collagen with anIC₅₀ of approximately 1.17 μg.mL⁻¹ (42 μM), 80% inhibition being reachedat a concentration of 5 to 10 μg.mL⁻¹ of _(m)scFv 9O12. This inhibitorycapacity was comparable to that observed for Fab 9O12 prepared afterpapain digestion of the parental IgG (2.1 μg.mL⁻¹; 15 nM).

The purified 9O12.2 scFv binds to human platelets and inhibitscollagen-induced platelet aggregation.

Binding of purified scFv to human platelets was measured by flowcytometry. A shift to the right of the peak indicates that the scFvbinds to human platelets in a dose dependant manner (FIG. 10). A nonrelevant purified scFv did not bind to platelets.

The effect of the 9O12.2 scFv on GPVI function was tested by measuringcollagen-induced platelet aggregation. For this purpose, the monomericforms of the scFv were purified by size exclusion gel chromatography.The 9O12.2 scFv (25 μg/mL) completely prevented collagen-inducedplatelet aggregation (FIG. 11) as did the 9O12.2 pFab used at the sameconcentration.

Platelet Aggregation Under Flow Conditions

In addition, the effects of _(m)scFv 9O12 on platelet adhesion andaggregation to collagen was investigated under arterial flow conditions,and compared to those of Fab 9O12 and an irrelevant scFv (see FIG. 12).Once again, platelet aggregation induced by collagen was inhibited. Inthe presence of the scFv or of the Fab, only isolated platelets wereobserved attached to the collagen fibers in agreement with previousresults (9; 41) and, in contrast to control conditions, no largeplatelet aggregates were observed.

Thrombin Generation

Since Fab 9O12 is known to inhibit thrombin generation at the surface ofcollagen-stimulated platelets, the effect of the purified _(m)scFv 9O12was tested using the thrombogram method (FIG. 13). _(m)scFv 9O12 and Fab9O12 reduced the thrombin peak to similar extents, and increased the lagpreceding thrombin generation, indicating that _(m)scFv 9O12 is asefficient as Fab 9O12 in inhibiting collagen-induced plateletprocoagulant activity.

1.2.2 First Humanized 9012.2 scFv (hscFv 9O12.2(1))

The inventors have then humanized the murine 9O12.2 scFv fragment asdescribed in paragraph 1.1.2. The nucleotide sequence and deduced aminoacid sequence of humanized VH and VL domains and of the first humanized9O12.2 scFv fragment (hscFv 9012.2(1)) are displayed in FIG. 14. ThishscFv 9012.2(1) fragment was then further characterized using affinitybinding to GPVI sepharose: The periplasmic fraction of recombinantbacteria was loaded onto a GPVI sepharose column. Retained proteins wereanalyzed by Western-Blot using an anti-c-myc IgG followed by anti-mouseantibody coupled to HRP and ECL revelation. A single band is detectedwith a molecular mass of 28.5 kDa (FIG. 15) as expected for thehumanized scFv.

The binding of hscFv 9O12.2(1) to GPVI-Fc was then analyzed and comparedto that of murine scFv 9O12.2 (mscFv 9O12.2). Purified humanized scFvand mscFv were injected on GPVI-Fc immobilized on a CM5 sensorchip.Results are displayed on FIG. 16 and show that hscFv 9O12.2(1) alsobinds to GPVI-Fc, with what appears to be a higher affinity than mscFv.

Finally, the binding of hscFv9O12.2(1) to human platelets was studied.Human platelets were incubated with bacterial periplasmic extracts.hscFv 9O12.2(1) bound to platelets was detected using FITC coupledanti-c-myc antibody. Binding to platelet were analyzed on XL EpicsCoulter Flow cytometer. An irrelevant scFv was used as a negativecontrol. Results are displayed in FIG. 17 and show a shift to the rightof the histogram for hscFv, which indicates that the humanized hscFv9O12.2(1) binds to platelet GPVI.

1.3 Conclusion

The results displayed above clearly show that despite essentialstructural modifications, the murine 9O12.2 scFv generated by theinventors from the 9O12.2 monoclonal antibody specifically binds tohuman GPVI with a high affinity. In addition, it inhibits GPVI bindingto collagen and prevents collagen-induced platelet aggregation, thusinhibiting GPVI function.

This scFv fragment thus displays all the advantages of scFv fragmentswhile retaining the affinity and inhibition capacity of correspondingFab fragments.

In addition, the inventors have also generated a first humanized versionof the 9O12.2 scFv fragment, which also binds to human GPVI with a highaffinity despite new essential structural modifications (hscFv9O12.2(1)).

This first humanized 9O12.2 scFv fragment (hscFv 9O12.2) further has theadvantage of a potentially minimally reduced immunogenicity.

This application thus describes for the first time a low molecularweight, low immunogenic GPVI ligand with a high capacity to inhibitplatelet aggregation, resulting in a very promising product for arterialthrombosis treatment.

Example 2 Synthesis and Activity of a Second Optimized Humanized SingleChain Variable Fragment hscFv 9O12.2(2) Directed Against HumanGlycoprotein VI

Production of the recombinant first humanized hscFv 9O12.(1) fragmentwas difficult, and so some refinements in the construct were called for.A second optimized humanized hscFv 9O12.(2) fragment was thus derivedfrom hscFv 9O12.(1).

2.1 Experimental Procedure

Construction of the Second Humanized scFv Fragment (hscFv 9O12.2(2))

We postulated that unforseen structural incompatibilities between theoriginal murine CDRs and the human acceptor frameworks could have led tomisfolding of the V-domains, and to the formation of insoluble inclusionbodies that remained sequestered in the bacterial cytoplasm. To getround these difficulties, some minor refinements were undertaken.

First, we observed that 9O12 CDR L1 loop is 5 residues longer than thatof 1VGE, and that 9O12 and 1VGE frameworks 1 and 2 have a low identityscore (48% and 73% respectively) (FIG. 19). This suggested that the 1VGE light chain FR1 and FR2 are not suitable for the correct folding ofCDR L1.

Extra FASTA searches were then performed using 9O12 VL FR1 and FR2. Anexcellent match was found with human 1×9Q for FR1 and FR2 (95.6 and 86.6identity scores respectively, and 100% similarities in both cases). Inaddition, loop L1 (CDR1) of the selected antibody 1×9Q was similar inlength to that of 9O12.

We therefore decided to preserve the original murine 9O12 VL FR1 and FR2in the novel humanized hscFv 9O12.(2) construction, since the sequencesof these frameworks fit well with another human antibody framework(1×9Q), the CDR L1 of which is exactly the same size as that of 9O12(see FIG. 19).

Other refinements were carried out on the basis of close inspection ofthe model. Indeed, we also retained in the final _(h)scFv 9O12 constructa very limited number of residues that could influence the ability ofCDR loops to adopt their conformation. Two critical areas werepreserved. The first one was the dipeptide LD at position 59-60 in VL.L59 (P in the human template), considered as potentially significant,since it is in the close vicinity of the residues of L CDR2. Although Land P are both hydrophobic, P has a cyclical side chain and is known toinduce specific effects on the protein backbone structure (MacArthur andThornton, 1999). In addition, we noticed that L occurs much less oftenthan P at this position (2%), and this may be indicative of a specificrole (Honegger & Pluckthun, 2001). The other unmutated murine residueswith no similarities to the human template (1VGE) were located in VH FR3(A_(H71), K_(H73), R_(H76), Kabbat numbering). Finally, only 5 murineresidues in VL and 10 in VH were maintained in the human frameworksselected for humanization.

The final construct is shown in FIG. 19. All the humanized hscFv9O12.(2) frameworks exhibit 100% similarity with human frameworks, apartfrom the VH and VL Frameworks 3 (90.62 and 93.75% respectively) (seefollowing Table 1).

TABLE 1 Identity and similarity scores of the 9O12 humanized V-domainframeworks with human antibodies frameworks used as template. x is thenumber of residues in the humanized FR that are identical to residuesfrom human FR. y is the total number of residues in the FR. FrameworksIdentity Similarities (FR) x/y % % V_(H) FR1 23/26 88.46 100 V_(H) FR214/14 100 100 V_(H) FR3 25/32 78.12 90.62 V_(H) FR4 11/11 100 100 V_(L)FR1 22/23 95.65 100 V_(L) FR2 13/15 86.60 100 V_(L) FR3 30/32 93.7593.75 V_(L) FR4 10/10 100 100

Globally, the 11 N-terminal residues from the murine VH FR3 werepreserved in the final construct, because they are clearly located closeto the flat part of the pocket in which the antigen is expected, and socould interact with it. Nevertheless, VH FR3 exhibits 25/32 residuesidentity with 1VGE. Only 3 residues of this framework (A_(H71), K_(H73),R_(H76), Kabbat numbering) had no similarity with 1 VGE. The 9O12 VL FR3was substituted for its 1VGE counterpart with the exception of tworesidues (L59P and D60S), essentially because L is not frequentlyencountered at this position, and D is an acidic residue.

Other Experimental Procedures

All other experimental procedures for characterizing the second hscFv9O12.(2) fragment were performed as described in Example 1.

2.2 Results

The production of hscFv9O12.2(2) was better than that of hscFv9O12.2(1).

In addition, the purified hscFv9O12.2(2) fragment conserved highaffinity for its target, as demonstrated by SPR analysis againstimmobilized GPVI (k_(on)=5.8×10⁴ M⁻¹s⁻¹, k_(off)=1.86×10⁻⁴ s⁻¹ anddissociation constant K_(D)=3.2 nM).

It was also able to bind to freshly prepared human platelets in flowcytometry, and the shift to the right of the fluorescence peak wasalways similar to that of cells labeled with the murine mscFv9O12 undersimilar experimental conditions (see FIG. 20B). Near-total inhibition ofhscFv9O12.2(2) binding was observed when platelets were pre-mixed withan excess of Fab 9O12. These results demonstrated that theaffinity-purified hscFv9O12.2(2) molecule retained the binding activity,affinity and specificity of the parent antibody for GPVI as well as fornative GPVI exposed at the surface of human platelets.

2.3 Conclusion

As for hscFv9O12.2(1), the main parameters that are usually affected byhumanization were well-preserved in hscFv9O12.2(2). Affinity-purifiedhscFv9O12.2(2) was fully functional, and had a high affinity for GPVI, amajor point for biological applications. FACS analysis also indicatedthat hscFv9O12.2(2) recognizes the same epitope on human platelets asmouse Fab 9O12, because its binding was specifically blocked in thepresence of a molar excess of Fab 9O12.

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The invention claimed is:
 1. A humanized single chain scFv antibodydirected against human glycoprotein VI, comprising or consisting of SEQID NO:28 or SEQ ID NO:47.
 2. The humanized single chain scFv antibody ofclaim 1, as a medicament.
 3. A pharmaceutical composition, comprisingthe humanized single chain scFv antibody of claim 1 and apharmaceutically acceptable carrier.
 4. A method for inhibiting plateletaggregation induced by collagen or treating thrombosis comprisingadministering to a subject in need thereof the humanized single chainscFv antibody of claim
 1. 5. An isolated nucleic acid encoding thehumanized single chain scFv antibody of claim
 1. 6. An isolated nucleicacid comprising or consisting of SEQ ID NO:29.
 7. An isolated nucleicacid comprising or consisting of SEQ ID NO:30 or SEQ ID NO:49.
 8. Anexpression vector comprising the isolated nucleic acid of claim
 5. 9. Anisolated host cell comprising the expression vector of claim
 8. 10. Amethod for preparing the humanized single chain scFv antibody of claim1, comprising: a) Culturing the host cell of claim 9, and b) Purifyingthe humanized single chain scFv antibody.